Substance P (SP) is a gut neuropeptide involved in physiological regulation and has also been implicated in gut inflammation. Physiological responses to SP have been shown to desensitize rapidly and extensively and SP receptors (SPR) appear to be up-regulated in inflammatory bowel disease. These observations suggest that the SPR is regulated but the characteristics and mechanisms of SPR regulation are largely unknown. This proposal describes studies designed to reveal mechanisms of SPR regulation. The specific aims are to (1) determine the mechanisms of SPR desensitization, (2) determine the mechanisms of SPR resensitization, and (3) determine the mechanisms of SPR up- and down- regulation. These goals will be achieved by testing several critical hypotheses. Specifically, the role of phosphorylation of SPR as a mechanism of desensitization will be assessed by experimental strategies such as inhibition of specific protein kinases, analysis of desensitization of mutagenized SPRs in which specific putative phosphorylation sites are deleted or substituted, and by analysis of the ability of the SPR to desensitize when SPR cells express a GRK inhibitor. For specific aim 2, the hypothesis that SPR resensitization requires dephosphorylation will be tested by examining the effects of specific cell-permeant protein phosphatase inhibitors and the hypothesis that receptor recycling back to the plasma membrane after agonist-induced endocytosis is also required for resensitization will be tested by examining the effects of agents that inhibit endosomal recycling. Finally, the ability of agonists, antagonists, and glucocorticoids to up- and down-regulate total cellular SPR levels and SPR mRNA levels will be assessed in order to begin to determine the mechanisms of cellular up- and down-regulation of substance P receptors. These studies will lead to insight into the normal mechanisms of SPR regulation and will suggest possible mechanisms of abnormal SPR regulation in inflammatory bowel disease.